Determination of analytes in particle-containing medium

ABSTRACT

Methods and compositions are provided for concentrating particles in a minute area on a solid surface. The method permits the detection of small amounts of analytes by providing for an observable signal in relation to the concentration of particles in the area.

BACKGROUND OF THE INVENTION

The clinical laboratory has become an increasingly important adjunct tomedicine, both in diagnosis and therapy. As the variety of situations inwhich determinations are desired have expanded, there has been anincreasing variety of approaches for measuring the substance ofinterest. There are many considerations involved in the development ofthe assay. One consideration is the simplicity of the protocol. The moremeasurements and steps that are required, the greater the likelihood forerror. A second consideration is the concentration range and absoluteamount to be measured. A third consideration is the nature of the sampleinvolved. A fourth consideration is the nature of pretreatments whichmay be required. A fifth consideration is the nature of interferingsubstances in the samples. A sixth consideration is the intendedenvironment in which the assay is performed and the technical skill ofthe persons who will perform the assay. This will also involve whetheran instrument is to be used or only a visual determination. Thus, eachnew development provides advantages which find particular applicationsas to analytes, preparation of reagents, nature of the users, and mannerof performance.

DESCRIPTION OF THE PRIOR ART

Anderson, Anal. Biochem. (1970) 38:175-189 describes the use ofcellulose wicks to monitor agglutination reactions.

SUMMARY OF THE INVENTION

Novel methods and compositions are provided for determining the presenceof analytes in a particle containing medium, where the analyte ofinterest may be bound or unbound to the particle in a sample. Bycontacting the assay medium with a bibulous material at a liquid airinterface, a small situs, usually a thin band or concentrated point, ofparticles can be obtained adjacent the interface, which site provides asignal which can be related to the presence of analyte in the sample.The particles include synthetic particles, cells, and immune complexaggregates. The size and nature of the particles, as well as the natureof the aqueous medium, can be used to modulate the formation of thesmall site.

.Iadd.BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a partial elevational view of a device in accordance with thepresent invention.

FIG. 2 is a cross-sectional view of the device of FIG. 1. .Iaddend.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The subject invention is based on the concentration of particles at asmall site, generally having one dimension less than about one mm wide,on a bibulous solid support in relation to the presence or absence of ananalyte in a sample. The site may be a point, a straight or curved band,or the like. The concentration of particles at the predetermined sitecan be used to provide a detectable signal at a site. The sample mayinfluence the concentration of the particles at the site and/or theproduction of a detectable signal. The detectable signal will bedetermined in a detection zone, which may or may not be theconcentration site.

The particles which are involved in the assay may be present in thesample, may be added as reagents or formed in situ. The nature of theparticle may vary widely, being naturally occurring or synthetic, beinga single material, a few materials, or a combination of a wide varietyof materials. Naturally occurring particles include nuclei, mycoplasma,plastids, mammalian cells, unicellular microorganisms, e.g., bacteria,etc. Synthetic particles may be prepared from synthetic or naturallyoccurring materials, such as metal colloids or latex particles made frompolystyrene polyacrylates or naturally occurring materials, such aspolysaccharides, e.g., agarose, or the like. Non-naturally occurringparticles may be varied depending upon the particular assay, theprotocol for the assay, or other considerations.

The size of the particles will vary widely, generally ranging from about0.05 to 100 microns, more usually from about 0.1 to 75 microns. Theparticles may be charged, either positively or negatively, may beamphoteric or lack any charge, being neutral. The presence or absence ofcharge will affect other parameters involved in the assay.

The means for detecting the detectable signal at or away from theconcentration site may or may not be an intrinsic property of theparticles. The particles may be labeled with a wide variety of materialswhich allow for detection, such as radionuclides, dyes, fluorescers,enzymes, or other convenient label providing for a detectable signal,either visually observable or detectable by instrumentation. The variouslabels would normally be covalently bonded to the particle, usinglinking arms as appropriate. The labels may be bound to the surface or,when feasible, extend throughout the particle.

Any convenient bibulous absorbent solid material may be employed whichallows for capillary transport of a liquid away from the interfacebetween the air and liquid. Various materials include paper, celluloseparticles, silica gel, cellulosic beads, glass fiber, filter paper, andthe like. The surface should be relatively smooth, so as to allow forthe formation of a concentrated particle site, for example, in the formof a sharp band or point. The size and shape of the bibulous materialmay be varied widely considering the purpose of the material. Namely,one wishes to form a fine band, curve, circle, line, or point byconcentrating small particles which are relatively uniformly dispersedin a liquid medium. The concentrating is best achieved by transporting arelatively large volume through a relatively narrow area. The bibulousmaterial may be shaped, therefore, as a narrow strip of from about oneto about five millimeters in width, a triangular shaped structure havinga rounded tip or a circular disc having a central small orifice or otherstructure. The size of the orifice may vary depending on whether theorifice extends solely through a non-bibulous support .Iadd.(such as,for example, depicted in FIGS. 1 and 2 wherein non-bibulous member 10has an orifice 12).Iaddend., leaving the bibulous member underneath theorifice intact, or the orifice extends through the bibulous memberunderneath .Iadd.(such as, for example, depicted in FIGS. 1 and 2wherein bibulous member 14 has orifice 16).Iaddend.. In the firstsituation, the orifice will generally be about 0.10 to 2 mm, usually0.25 to 1 mm. In the second case, the orifice will generally be lessthan one millimeter, ranging from about 0.1 to 0.5 mm. In each case, thebibulous material will usually have a support which provides structuralstrength. The non-bibulous material may be a water impermeable layer orcoating.

The liquid medium will normally be an aqueous medium, which may havefrom about 0-40 volume percent of a miscible solvent such as alkanols,ethers, sulfoxides, amides, etc., generally ranging from about 1 to 6carbon atoms.

In performing the subject invention, one usually wishes to concentrateor collect particles at the air-liquid interface depending upon thepresence or absence of a predetermined condition. The condition will bethe presence in the sample, above a predetermined amount, of an analytewhich is a member of a specific binding pair.

By appropriate choice of conditions in the aqueous medium, one canmodulate the size of particles which will concentrate at the air-liquidinterface as contrasted with following the solvent front, so that no orfew particles collect at the air-liquid interface, resulting in theabsence of an observable site.

The choice of conditions will vary, with the nature of the particle, asto size, charge, polarity or other property which affects the repulsionor attraction of the particles to each other. In order to form theconcentrated particle site, one wishes to distinguish between particlesof a particular size or between different sized particles. In the formersituation, the method serves to concentrate particles present in themedium above a predetermined size. In this situation, the particles donot undergo a change in size distribution as a result of the presence ofan analyte. In the latter situation, the presence of an analyte willresult in the binding together of particles, where the original sizedparticles would follow the solvent front, while particles which arebound together will remain on the bibulous surface at the air-liquidinterface. Therefore, the conditions will be chosen so that particles ofabove a certain size will be retained at the air-liquid interface, whileparticles smaller than that size will travel away from the air-liquidinterface.

Factors that affect the size of the particle which will migrate involverepulsive and attractive forces, which can be influenced by pH and ionicstrength. Where the particles are heavily charged with the same charge,at a relatively high ionic strength, the charge will be neutralizedwhich will allow the particles to aggregate or band together. Where theparticles have acidic or basic groups, a pH can be chosen, to reduce thenumber of charges present on the particle. Conditions can be chosen sothat the particles will not aggregate unless a binding means isprovided. The binding means will be a member of a specific binding pair,which is polyvalent and is, therefore, capable of binding to at leasttwo particles.

The pH will vary with the nature of the particle. For basic particles,one may induce repulsive charges by lowering the pH, while for acidicparticles, one may induce repulsive charges by raising the pH. The pHwill be chosen so as to maintain mild repulsion between particles, so asto encourage the transport of the particles, except where a plurality ofparticles are joined together.

Ionic strength may also be used in a similar fashion to modulaterepulsion. By reducing or raising the ionic strength, one may modulatethe repulsive effect between particles, so that at low ionic strength,one enhances the repulsive effect, while at high ionic strength, onereduces the repulsive effect between charged particles. Therefore,depending on the nature of the particles, one will modify the conditionsof the medium. The various conditions will be optimized depending on thenature of the system.

The pH which is employed will generally be in the range of about 2 to12, with the range varying from about 3 to 7 for positively chargedparticles and from about 6 to 11 for negatively charged particles. Theionic strength will generally vary from about 10⁻¹ to 10⁻⁴. One mayoptimize these two parameters empirically depending upon the size andnature of the particles involved.

A further factor is the inclusion of surfactants in the assay medium.The surfactants aid in the migration of the particles through thebibulous support. By modifying the surface tension, migration of theparticles may be enhanced or diminished. The surfactants may be anionic,cationic or non-ionic, preferably non-ionic or combinations of non-ionicand anionic. Surfactants will be used in minor amount, generally beingpresent in from about 0 to 2 vol % of the assay medium, more usuallyfrom about 0.005-1.5 vol %, and preferably from about 0.01 to about 1vol %. Various surfactants may be used, such as Tween 20, QS44, PEG1500, etc.

Other factors may also be employed to affect properties of the assaymedium. Chaotropic or antichaotropic agents may be employed.Illustrative chaotropic agents include fluoride ion and polyethyleneglycol. Illustrative antichaotropic agents include trichloroacetate,thiocyanate and dextran. Agents to modify the viscosity of the mediummay be employed. Elevated or reduced temperatures may also findapplication.

Method

As indicated previously, the subject method is predicated on eitherconcentrating particles over a predetermined size at a localized site orbeing able to distinguish two sets of particles: (a) Particles whichmigrate with the solvent away from the air-liquid interface and (b)particles which form a localized site at the air-liquid interface. Thelatter situation will be the more common one.

In the former case, the primary function is concentrating particleswhich are present in a dilute solution. This situation may beexemplified by a mixture of particles where only a small percentage maybe the particles of interest. For example, a clinical sample which has aheterogeneous population of cells, where one wishes to determine thepresence of a particular species or strain. By employing a labeledantibody in the medium, one could rapidly tag any cells of interest. Thepresence of the tag at the localized site would be diagnostic of thepresence of the particular cells. Any unbound label would follow thesolvent front, minimizing any background. The absence of an observableband would indicate the absence of the cells of interest in the sample.

A particle is employed which migrates under the conditions of the assay,but in the presence of analyte in the medium, can be inhibited frommigrating or permitted to migrate. In this way, one can relate thepresence of a detectable signal at a localized site on a bibuloussurface to the presence of the analyte of interest in the assay medium.

The concentration of particles at the localized site is achieved byproviding bridges between particles of specific binding members.Specific binding members can be broken up into two primary groups: (1)ligands and receptors; and (2) complementary polynucleotides. Theligands and receptors involve organic molecules, where the ligand is anymolecule for which a receptor is available or can be made. The ligand ischaracterized by having a polar and spatial organization which binds toa reciprocal or homologous receptor. The receptor is conventionally amacromolecule which has a structural organization complementary to theligand so as to have a high avidity for the particular structure of theligand to provide for a specific binding complex. Conventional receptorsinclude antibodies and fragments thereof, enzymes, naturally occurringreceptors, and the like.

In the subject invention, the ligands may be haptens or antigens, butwhere the ligand is monovalent and has to serve as a bridge, it will beprovided in a polyvalent form. The polynucleotides may be DNA or RNA,where the bridge will have a sufficiently extended complementarysequence, for example, by repeating the same sequence to allow bindingto two different fragments of complementary polynucleotide sequences.

In carrying out the method, one combines the sample, the assay mediumhaving the appropriate conditions for the particles, particles, ifparticles are to be added, and the bridging system for bridging theparticles. Depending upon the manner in which the localized site is tobe detected, a signal producing system may also be involved, where oneor more labels are provided bound to members of the bridging system orto binding members which bind to the analyte. By having two differentbinding members involved in the production of a detectable signal, onecan provide for detection of an analyte having two different reciprocalbinding members.

After combining the sample with the bridging members and any signalproducing members, as appropriate, in an appropriate assay medium, onethen contacts a small portion of the bibulous member with the assaymedium, where a major portion of the bibulous member does not contactthe assay medium and may act as a wick or well for absorbing liquid, forexample, by capillary or wicking action, so as to draw liquid throughthe area at the air-liquid interface.

After sufficient time to allow for a sufficient proportion of the assaymedium to be absorbed by the bibulous member, so that concentrations ofthe particles at the localized site can form, as appropriate, contactwith the assay medium may be terminated. Depending upon the signalproducing system, the presence of the site may be determined oradditional members of the signal producing system may be added to thearea at which the site may have formed to provide a detectable signal.If a quantitative result is desired, the detectable signal may bemeasured by any convenient means.

To further illustrate the subject invention, the following illustrativeexamples will be described. In an assay for a hapten, one could providecolored beads to which a hapten is covalently bonded by an appropriatelinking arm. The assay conditions and size of beads, as well as thenature of the bibulous member, would be chosen, so that the individualbeads would migrate with the solvent, and no band would be observed. Onecould then combine the sample and antibodies, so that at a concentrationof interest of the analyte, a major proportion of the antibody siteswould be filled by the available hapten.

After sufficient time for binding to occur, one could then add theparticles to the assay medium and incubate a second time to allow forbinding of any available antibody binding sites to the hapten on thecolored beads. In the absence of hapten in the sample, there would be asubstantial amount of bridging between the beads by the antibodies.

One would then contact the bibulous member with the assay medium andallow a sufficient amount of the assay medium to be wicked into thebibulous member so that a substantial number of particles may traversethe air-liquid interface adjoining the bibulous member. Based on thecolor of the beads, one would observe a sharp, distinct band in theabsence of hapten and substantially no beads in the presence of hapten.Where one is interested in a range of concentration of the hapten, acontrolled amount of the assay medium would be absorbed by the wick andthe concentration of particles determined by appropriatespectrophotometric measurements, e.g., using a reflectometer. Thisresult could be compared with the result observed with a sample having aknown amount of the hapten.

A second illustrative example is the determination of an antigen. Beadscould be provided which are labeled with antibodies to the antigen andan enzyme, for example, horseradish peroxidase. One would combine thebeads under conditions where individual beads would migrate with thesolvent, but linked beads would remain at the juncture of the air-liquidinterface. One would combine the beads with the sample and incubate themixture for sufficient time to allow the antigen to bind to theantibodies to provide bridges between the beads. One would thenintroduce the bibulous member as before and allow a sufficient amount ofthe liquid medium to be wicked by the bibulous member.

After a sufficient amount of the medium had been wicked through thebibulous member to allow for the formation of a narrow band or point,the bibulous member would be removed from the assay medium and placed ina development solution containing hydrogen peroxide and a substrate forhorseradish peroxidase, which upon oxidation forms a color, desirablyforms an insoluble dye, which precipitates onto the beads. In thismanner, the band or point would become visually observable whereparticles are present to which horseradish peroxidase has become boundor retained.

A third illustrative example is where one is interested in a particularbacterial strain. In this method one might choose a bibulous membercircle mounted on a non-bibulous plastic support having a centrallylocated orifice of about 0.5 mm dia. A swab is taken of a clinicalsample and dispersed in PBS-0.05% Tween 20. To the dispersion is added amonoclonal antibody conjugated to an enzyme and the mixture isincubated. A few drops of the sample are placed over a device comprisinga lower bibulous disc bonded to an upper hydrophobic plastic disc havinga central orifice of about 0.5 mm dia. The liquid passes through theorifice and spreads evenly outward from the orifice with the organismsconcentrated to the spot on the bibulous member underneath the orifice.The monoclonal antibody-enzyme conjugate will bind to any organismswhich have the reciprocal epitopic site. Excess monoclonalantibody-enzyme will wick away so that only specifically bound enzymewill remain as the spot. To ensure removal of residual enzyme which isnot specifically bound, a few drops of the PBS-Tween 20 solution may beplaced over the orifice and allowed to wick. About 0.05 ml of adeveloper solution is then added, e.g., H₂ O₂ +4-chloronaphthol with HRPas the enzyme and the solution allowed to wick. Color will indicate thepresence of the organism.

A large number of patents have been issued which describe a wide varietyof labels which have found use in diagnostic assays. Various protocolscan be developed where these labels may be used with advantage.Illustrative of such patents are U.S. Pat. Nos. 3,850,752; 4,255,329;4,233,402; and 4,208,479.

For performing the method, kits can be provided where the variousreagents are combined in predetermined amounts in combination withvarious ancillary materials for combination with the sample. In view ofthe wide spectrum of protocols and reagents, a wide variety of kits maybe prepared. For the most part, where the method involves the additionof particles, the kits will involve particles which have a member of aspecific binding pair substantially irreversibly bound to the particle,either covalently or non-covalently. Also, there may be a label bound tothe surface of the particle or dispersed therein, particularly a dye,which may be colored in the visible range of fluorescent. In someinstances, the particle may also be labelled with an enzyme.

Where particles are not to be included, the reagents will normallyinvolve labelled receptors or ligands, where the labels provide for adetectable signal and may provide for the inhibition of migration of theparticles present in the assay medium. In addition to the labelledreagents, there will be ancillary reagents such as buffers, stabilizers,detergents, and as appropriate substrates for enzymes, bulking agents,and the like. Also included in such kits would be the wickingmaterial--prepared as strips or discs--with precut orifices, etc.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

In the following experiment, a variety of beads of different colors andsizes were combined to demonstrate that one could achieve bands ofdifferent colors depending upon the sizes of the beads and their colorand the effect of the combination of the beads in a band. The protocolwas to combine 0.05 ml of each 2% bead preparation plus 20 ml of a 1%solution of normal sheep serum in PBS containing 0.05% QS44 and 0.05%Tween 20. The paper which was employed was triangular-shaped Milliporemembrane paper with a rounded tip, where the tip was inserted into theassay medium. This shape provides for enhanced concentration of theparticles, while providing a large wicking reservoir.

The following table indicates the beads that were employed, the expectedcolor and the observed color.

                  TABLE 1                                                         ______________________________________                                        Bead Color                                                                    Red        Blue   Yellow    Color                                             Size, μ          Expected  Observed                                        ______________________________________                                        1.    .15      .5     .5      green   green                                   2.    .5       .25    .5      orange  orange                                  3.    .5       .5     .25     purple  purple                                  4.    .15      .25    .5      yellow  yellow                                  5.    .5       .25    .25     red     red                                     6.    .15      .5     .25     blue    blue                                    7.    .5       .5     .5      brown   brown                                   8.    .15      .25    .25     white   No color                                ______________________________________                                    

The colors were predicted based on the 0.5 micron particles beingretained on the surface adjacent the interface, while the small particlemigrated with the solvent front; thus, the complementary color of thelarger beads is seen.

In the next experiment, an assay was performed to detect the presence ofStreptococcus pyogenes (Lancefield Group A) in a mixture of Group A andGroup B. The assay medium was prepared by removing cells of the twoorganisms from agar growth by loop and suspending them in 1 ml of PBSplus 0.05% Tween 20. A mouse monoclonal antibody against Group A(anti-A) at about 5 mg/ml was employed. Also employed was goatantibodies against mouse IgG (G-anti-IgG) which was conjugated tohorseradish peroxidase. As a developing solution, a solution wasemployed containing 200 μg/ml of sodium 4-chloro-2-naphthol, 50 mMglucose, 2 mg/ml bovine serum albumin and excess hydrogen peroxide. Theprotocol was to combine 100 ml of the organism solution (about 10⁷cells/ml), 20 μl of the antibody-horseradish peroxidase conjugate and 20μl of the anti-A, incubate the mixture and then wick 50 ml on acellulose strip (cellulose strips used for tlc). The strip was thenplaced in the substrate solution and a dark band formed indicating thepresence of Group A organisms.

A similar experiment was carried out, where enzyme channeling wasinvolved, demonstrating a rapid antibiotic sensitivity assay.

Group A strep. pyogenes (0.2 ml of 10⁸ cells/ml) were incubated for 23/4hrs. at 37° C. in the presence or absence of penicillin at 2 units/ml.This solution was then combined with 0.8 ml of phosphate buffered saline(0.02 ml phosphate plus 0.05% Tween 20 - pH 7.2) followed by theaddition of 0.01 ml of a monoclonal anti-Group A antibody conjugated tohorseradish peroxidase (HRP). 0.2 ml of this mixture was wicked for 7min. with a cellulose TLC paper strip. The wick was then transferredinto developer (0.2 ml of the above-described 4-chloronaphthyl substratesolution plus 0.5 μl glucose oxidase). The wick was developed for 30min.

RESULTS

Bacterial cells exposed to the penicillin formed a less intenseblue-purple band than did the unexposed cells. The difference was easilydetectable by the eye.

It is evident from the above results, that a simple rapid method isprovided for detecting the presence of an analyte. The method can bequalitative or quantitative and can be used with a variety of protocolswhich can be adapted to particular samples. Furthermore, simpleequipment is employed and the results can be obtained by visualobservation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for detecting the presence of a memberof a specific binding pair ("sbp member") in a .[.liquid assay medium.]..Iadd.sample.Iaddend., said specific binding pair consisting of ligandand homologous receptor, where said method involves particles to whichare bound at least one member of a specific binding pair, a solidbibulous member and a signal-producing system which involves at leastone label which is bound to said particles or an sbp member,said methodcomprising: combining in an aqueous assay medium, .[.said.]. a sample.Iadd.suspected of containing an sbp .Iaddend.member and at least one.Iadd.of the group consisting .Iaddend.of said particles and .Iadd.said.Iaddend.labeled sbp members, with the proviso that particles are addedwhen said sample lacks particles having an sbp member; under conditionswhere particles only within a predetermined size.[.,.]. range and chargewill concentrate in an area on said bibulous member adjacent theair/liquid interface, when said bibulous member is contacted with saidassay medium; contacting said bibulous member with said assay mediumwhereby said assay medium is wicked past said area and particles withinsaid predetermined size .Iadd.range .Iaddend.and charge concentrate at asmall site in said area; and detecting the signal as a result of saidsignal-producing system, wherein said signal is related to the amount oflabel in said area and the amount of label in said area is related tothe amount of said sbp member in said sample.
 2. A method according toclaim 1, wherein said sample has particles within said predeterminedsize .Iadd.range .Iaddend.to which are bound an sbp member.
 3. A methodaccording to claim 1, wherein said sample has particles below saidpredetermined size .Iadd.range .Iaddend.to which are bound an sbp memberand said label is joined to a polyvalent homologous sbp member.
 4. Amethod according to claim 1 wherein said particles are labeled with amember of said signal producing system.
 5. A method for detecting cellsthat have at least one predetermined determinant site, where said methodinvolves labeled receptors which bind to said determinant site or toreceptors binding to said determinant site, and a solid bibulous member,where the label of said labeled receptor by itself or in combinationwith other members of a signal producing system produces a detectablesignal, said method comprising:combining in an aqueous assay medium, asample suspected of containing said cells, labeled receptors to saiddeterminant site or receptors for said determinant site and labeledreceptors for said receptors, under conditions where individual cellsmigrate along a bibulous member away from an air-liquid interface whensaid medium is contacted with said bibulous member, but receptor linkedcells concentrate at .[.the.]. .Iadd.an .Iaddend.area of .Iadd.said.Iaddend.bibulous member at the air-liquid interface; contacting saidbibulous member with said assay medium, whereby linked cells concentratein said area; and detecting the signal as a result of said signalproducing system wherein said signal is related to the amount of labelin said area.
 6. A method according to claim 5, wherein said cell is abacterial cell, fungal .Iadd.cell.Iaddend., protozoan.Iadd.cell.Iaddend., or other parasitic or infectious agent.
 7. A methodaccording to claim 5, wherein said label is an enzyme.
 8. A methodaccording to claim 5, wherein said bibulous member has a small orificesurrounded .[.on one side of said bibulous member.]. with a waterimpermeable layer and said assay medium is contacted with said bibulousmember at said orifice.
 9. A method according to claim 5, wherein saidbibulous member is supported by a plastic support having a centrallylocated orifice. .Iadd.10. A method according to claim 1 wherein aftersaid contacting of said bibulous member with said assay medium, saidarea on said bibulous member is contacted with an aqueous mediumcontaining a labeled sbp member when said labelled sbp member is notincluded in said assay medium. .Iaddend. .Iadd.11. A method according toclaim 1 wherein said contacting is carried out by introducing a portionof said bibulous member into said assay medium. .Iaddend. .Iadd.12. Amethod according to claim 1 wherein said contacting is carried out byintroducing a portion of said assay medium onto said bibulous member..Iaddend. .Iadd.13. A method according to claim 1 wherein said combiningis carried out by combining said sample in an aqueous medium with saidparticles and said contacting is carried out by contacting said assaymedium with a portion of said bibulous member. .Iaddend. .Iadd.14. Amethod for detecting the presence of an analyte, said methodcomprising:combining a medium suspected of containing said analyte,which is a member of a specific binding pair (sbp), with particleshaving bound thereto an sbp member reciprocal to said analyte,contacting said medium with an area of a bibulous member underconditions where said medium wicks away from said area and at least aportion of said particles concentrate in a localized site on saidbibulous member, and examining said site to determine the presence ofsaid analyte. .Iaddend. .Iadd.15. The method of claim 14 wherein saidparticles have a label. .Iaddend. .Iadd.16. The method of claim 14wherein said examining is carried out by (1) contacting said site with amedium containing a labeled receptor for said analyte and (2)determining the presence of said labeled receptor at said site todetermine the presence of said analyte. .Iaddend. .Iadd.17. The methodof claim 14 wherein said examining is carried out by (1) contacting saidsite with a medium containing a labeled receptor for said reciprocal sbpmember and (2) determining the presence of said labeled receptor at saidsite to determine the presence of said analyte. .Iaddend. .Iadd.18. Themethod of claim 14 wherein said site is examined by contacting said sitewith members of a signal producing system and then examining said sitefor the presence of a detectable signal to determine said analyte..Iaddend. .Iadd.19. The method of claim 14 wherein said medium wicksaway from said area by migrating outward along said bibulous member..Iaddend. .Iadd.20. A method for detecting the presence of an epitopicsite on particles, said method comprising:contacting a spot on abibulous member with a medium containing a labelled antibody reciprocalto said epitopic site and particles suspected of having said epitopicsite under conditions where excess of said labelled antibody will wickaway from said spot; and detecting the presence of said labelledantibody at said spot. .Iaddend. .Iadd.21. The method of claim 20wherein said antibody is labelled with an enzyme or a fluorescer..Iaddend. .Iadd.22. A method for detecting the presence of an analyte,said method comprising: providing in combination an aqueous mediumsuspected of containing said analyte, which is a member of a specificbinding pair (sbp), particles having bound thereto an sbp memberreciprocal to said analyte, and an area of a bibulous member underconditions where said medium wicks away from said area and at least aportion of said particles concentrate in a localized site on saidbibulous member, and examining said site to determine the presence ofsaid analyte. .Iaddend. .Iadd.23. The method of claim 22 wherein saidparticles have a label. .Iaddend. .Iadd.24. The method of claim 22wherein said examining is carried out by (1) contacting said site with amedium containing a labeled receptor for said analyte and (2)determining the presence of said labeled receptor at said site todetermine the presence of said analyte. .Iaddend. .Iadd.25. The methodof claim 22 wherein said examining is carried out by (1) contacting saidsite with a medium containing a labeled receptor for said reciprocal sbpmember and (2) determining the presence of said labeled receptor at saidsite to determine the presence of said analyte. .Iaddend. .Iadd.26. Themethod of claim 22 wherein said examining is carried out by (1)contacting said site with members of a signal producing system and then(2) determining the presence of a detectable signal at said site todetermine the presence of said analyte. .Iaddend. .Iadd.27. The methodof claim 22 wherein said medium wicks away from said area by migratingoutward along said bibulous member. .Iaddend. .Iadd.28. A device forconducting an assay, said device comprising a non-bibulous member havingan orifice and serving as a support for a bibulous member, said bibulousmember having an orifice that extends through said bibulous member, saidorifices corresponding in location. .Iaddend. .Iadd.29. The device ofclaim 28 wherein said orifice of said bibulous member and said orificeof said support are centrally located. .Iaddend. .Iadd.30. The device ofclaim 28 wherein said bibulous member is a circular disc. .Iaddend..Iadd.31. A kit comprising in packaged form:a device comprising anon-bibulous member having an orifice and serving as a support for abibulous member such that, when said device is used in conducting anassay, liquid containing particles applied to said bibulous memberthrough said orifice migrates away from point of application and atleast a portion of the particles present in said liquid concentrate atsaid joint of application and in a separate container particles whichhave bound irreversibly thereto a member of a specific binding pair..Iaddend. .Iadd.32. The kit of claim 31 which further comprises in aseparate container a member of a signal producing system. .Iaddend..Iadd.33. A method for detecting the presence of a member of a specificbinding pair ("sbp member") in a sample, said specific binding pairconsisting of ligand and homologous receptor, where said method involvesparticles to which are bound at least one member of a specific bindingpair, a solid bibulous member and a signal-producing system whichinvolves at least one label which is bound to an sbp member, said methodcomprising: combining in an aqueous assay medium, a sample suspected ofcontaining an sbp member and said particles and said labeled sbp memberunder conditions where said particles concentrate in an area on saidbibulous member adjacent the air/liquid interface, when said bibulousmember is contacted with said assay medium; contacting said bibulousmember with said assay medium whereby said assay medium is wicked pastsaid area and particles concentrate at a small site in said area; anddetecting the signal as a result of said signal-producing system,wherein said signal is related to the amount of label in said area andthe amount of label in said area is related to the amount of said sbpmember in said sample. .Iaddend. .Iadd.34. The method of claim 33wherein said label is an enzyme. .Iaddend. .Iadd.35. The method of claim33 wherein said sbp member in said sample is an antigen, said sbp memberbound to said particles is an antibody for said antigen and said sbpmember bound to said label is an antibody. .Iaddend. .Iadd.36. Themethod of claim 33 wherein said sbp member in said sample is a hapten,said sbp member bound to the particles is an antibody for said haptenand said sbp member bound to said label is said hapten. .Iaddend..Iadd.37. A method for detecting the presence of an analyte, said methodcomprising: combining a medium suspected of containing said analyte,which is a member of a specific binding pair (sbp), with particleshaving bound thereto an sbp member reciprocal to said analyte and with alabeled sbp member wherein the amount of labeled sbp member which bindsto said particles is related to the amount of analyte in said medium,contacting said medium with an area of a bibulous member underconditions where said medium wicks away from said area and saidparticles concentrate in a localized site on said bibulous member, andexamining said site for the presence of labeled sbp member to determinethe presence of said analyte. .Iaddend. .Iadd.38. The method of claim 37wherein said site is contacted with members of a signal producing systemand then is examined for the presence of a detectible signal todetermine the analyte. .Iaddend. .Iadd.39. The method of claim 37wherein said medium wicks away from said area by migrating outward alongsaid bibulous member. .Iaddend. .Iadd.40. A method for performing animmunoassay for an analyte in a sample, which method comprises providinga chromatographic medium, forming a reaction mixture containing aspecific binder for the analyte, said specific binder being immobilizedby attachment to a particulate solid phase, and a labeled reagent partlyin a form which is mobile on said medium and partly in the form of alabeled analyte/specific binder reaction product insolubilized so as tobe immobile on the medium, the proportions of the two forms beingdetermined by the amount of analyte in the sample, applying the reactionmixture to a spot on the chromatographic medium, causing the mobile formof the labeled reagent to migrate from the spot, and thereafter,observing one or both of the mobile and immobile, insolubilized forms ofthe labeled reagent. .Iaddend. .Iadd.41. The method as claimed in claim40, wherein the chromatographic medium is in sheet form, with thereaction mixture being applied to a spot on the sheet and the mobileform of the labeled reagent being caused to migrate radially from thespot. .Iaddend. .Iadd.42. The method as claimed in claim 40, whereinmigration is assisted by subsequent application to the spot of a solventwhich causes mobile species, but not immobile species, to migratethrough the chromatographic medium. .Iaddend. .Iadd.43. The method asclaimed in claim 42, wherein the solvent is an aqueous liquid. .Iaddend..Iadd.44. The method as claimed in claim 42 wherein the reaction mixtureis formed by causing the analyte in the sample, and a labeled version ofthe analyte, to compete for reaction with a limited amount of a specificbinder for the analyte, the analyte/specific binder complex beingrendered insoluble. .Iaddend. .Iadd.45. The method as claimed in claim44, wherein the analyte/specific binder complex is rendered insoluble byreaction of the specific binder with its antibody, said reaction beingeffected before, during and after incubation with the analyte. .Iaddend..Iadd.46. The method as claimed in claim 40, wherein the reactionmixture is formed by causing the analyte in the sample to bind to anexcess of an immobilized specific binder and a labeled specific binderfor the analyte is caused to bind to vacant binding sites of theanalyte. .Iaddend.